Although cigarette smoking continue to be the most popular and persistent method of recreational drug use in the U.S., relatively little is known about the neurochemical effects of nicotine in the brain or about the neurochemical consequences of chronic nicotine exposure. The overall hypothesis to be tested by the studies outlined in this proposal is that the functioning of the nicotinic cholinergic receptor (nAChR) in the CNS of the rat is altered by chronic exposure of the animal to nicotine. We will carry out studies to explore this hypothesis and to determine the mechanism(s) involved in the effects of nicotine that we identify. The specific aims of the studies outlined in this proposal are (1) to explore using neurochemical methods, the ionic events that follow nAChR activation in synaptosomes; (2) to determine whether the number of nAChR is changed following chronic exposure of rats to nicotine using an approach that does not rely on agonist binding to the receptor; (3) to determine whether the functional status of the nAChR ionophore is altered following chronic treatment with nicotine; (4) to explore the mechanism involved in the effects of in vivo nicotine on synaptosomal membrane potential; (5) to explore the effects of chronic nicotine on synaptosomal calcium influx (6) to separate nAChR containing synaptosome and determine whether they are selectively affected by nicotine exposure or if a large pool of synaptosome will be loaded with PTSA and the entry of cations in response to nicotine and other agonist will be measured as a decrease in fluorescence in the presence of thallium. We will explore the effects of nAChR activation on intrasynaptosomal free calcium concentrations using either Fura-2 and conventional spectrofluorometric approaches or Indo-1 with flow cytometry. Our method for labeling, quantifying and sorting synaptosome with nAChR will involve coupling a fluorochrome to a commercially available antibody that recognizes the Beta2 subunit of the CNS nAChR (mAb270). We will use the technique of flow cytometry coupled wit fluorescence activated cell sorting to determine the concentration of antibody (and hence the concentration of nAChR) in synapto-somes from nicotine exposed animals. Furthermore, we will use the cell sorting capabilities of the flow cytometer to isolate a synaptosomal fraction that is enriched in nAChR-containing terminals. It is anticipated that the successful completion of these studies will enhance our understanding of the mechanism of action of nicotine, give insight into the compensatory mechanisms involved in the development of tolerance and provide general information regarding regulatory processes that operate in the nerve terminal.